Project 1 - Abstract The formation of Factor VIII (FVIII) inhibitor is a main complication of the treatment of hemophilia A (HA) and it is associated with increased morbidity and mortality. The overall goal of this project is to gain insights into why FVIII is immunogenic by understanding how FVIII is targeted by the humoral immune system. We hypothesize that the FVIII-specific humoral immune response is composed of a diverse and dynamic antibody repertoire. We assembled a group of investigators from a variety of complementary scientific background that together will overcome major gaps in knowledge on FVIII immunogenicity and B cells. We will employ innovative and state-of-art technologies to broad our current understanding of the underlying mechanism of inhibitor formation to FVIII. We propose the following aims: Aim 1: Define the clonal repertoire and epitope specificity of circulating anti-FVIII antibodies in patients with HA. Taking a novel immunomics approach, we will, for the first time, characterize in detail the FVIII epitopes recognized by FVIII-specific antibodies as well as clonal relationships of the related B cells in HA patients with inhibitors. Aim 2: Define the FVIII antibody repertoire and its dynamics in canine HA inhibitor models. Utilizing antigen phage display for the first time in canine disease models, we will longitudinally characterize the FVIII-driven humoral immune response, which will provide insight into the intrinsic immunogenicity of FVIII. Results from this work will be compared to data from Aim 1 to further validate the HA dog model. Aim 3: Investigate the role of B cell survival cytokines in inhibitor formation, epitope diversity and eradication. Emerging evidence implicates B cell survival cytokines, including BLyS, in immune-mediated diseases. Our preliminary data suggests that BLyS may have a role in FVIII inhibitors. Herein, we will comprehensively evaluate the contribution of BLyS to FVIII immunogenicity through a series of studies in human and murine HA models. Together these studies will provide insights on the humoral immune system in FVIII immunogenicity, including establishing a framework for novel therapeutic approaches and testing the validity of small and large preclinical models.